There is an increasing understanding that epigenetic changes contribute to cancer development. Because epigenetic changes are by definition reversible, and because many epigenetic modifying enzymes are highly amenable to drug discovery, there is an explosion in interest in developing epigenetic-targeted therapies. Despite being highly druggable, a major impediment to clinical translation of epigenetic therapies is the lack of understanding as to the clinical diseases to which these drugs should be applied. We are therefore at a crossroad in which multiple entities within academia and industry are actively developing drugs targeting epigenetic modifying proteins, but the diseases to which they should be applied are as yet poorly defined. We hypothesize that a comprehensive and systematic assessment of which epigenetic regulating proteins are critical for in vivo tumor growth will help define the epigeneti targets appropriate for therapeutic application in specific cancer sub-types. In this proposal, we will use pooled shRNA analysis to systematically determine, which epigenetic-modifying proteins are critical for the in vitro and in vivo growth of Ewing sarcoma cells. In the first Specific Aim, we have used gene annotation and protein domain analysis to identify 449 gene products that have known or putative epigenetic regulatory function (the epigenome). We will package a shRNA library composed of 3040 shRNAs targeting the epigenome and controls. In the second Specific Aim, we will use this pooled library to transduce 4 Ewing sarcoma cell lines that have been iteratively selected for high tumorigenicity. Next-generation sequencing will be used to determine the frequency of all shRNAs in an input sample compared to samples selected after in vitro growth in cell culture and in vivo growth as xenograft tumors. An analysis pipeline will identify genes products essential for in vitro and in vivo tumor cell growth. In the third Specific Aim we will fully assess the cellular effects associated with loss o function of specific high priority epigenetic regulatory proteins identified in the preceding Aims, and will assess therapeutic targeting in established Ewing sarcoma xenografts. Together, the overall goal of these studies is to systematically determine which epigenetic regulatory proteins are required for Ewing sarcoma growth and to achieve in vivo proof-of-concept for therapeutic targeting of Ewing sarcoma with epigenetic therapies. Success in these studies will establish a paradigm that can be readily applied to almost all other cancer sub-types.